This invention relates to an isolated plant-gene promoter and to methods for using such a promoter. More specifically, the promoter was obtained from a gene encoding a metallothionein-like protein.
Metallothionein-like Genes and Their Expression Patterns
Genes encoding Metallothionein-like proteins (i.e., xe2x80x9cmetallothionein-like genesxe2x80x9d or xe2x80x9cMT-like genesxe2x80x9d) can be categorized into two classes based on the pattern of cysteine distribution within their predicted translation products (Robinson et al., Biochem. J. 295:1-10, 1993). Class I MT-like proteins contain two cysteine-rich domains, as found in animal metallothioneins, and class II MT-like proteins include an additional cysteine-rich domain within the protein. Class I MT-like proteins are further classified into three types (types 1-3) distinguished by the characteristics of their cysteine-containing domains. Each type of MT-like protein also has similar amino acid sequences within spacer regions between the cysteine-rich domains.
To date, Arabidopsis is the only plant species in which metallothionein-like genes of all categories (classes I, II, and types 1-3 of of class I) have been identified. The presence of representatives of each category within a single species (e.g., Arabidopsis) or within closely related species (e.g. wheat, barley, and rice) is significant, as it suggests that plant MT-like proteins may have distinct functions in relation to their structure, patterns of expression, and response to stresses.
The published data on expression of various MT-like genes from a variety of plant species, is summarized in Table 1.
From the data in Table 1, it is clear that each MT-like gene type exhibits characteristic developmental and tissue-specific expression patterns. The expression of class II MT genes, such as for the wheat and maize EcMT, is restricted to immature embryos (Kawashima et al., Euro. J. Biochem. 209:971-976, 1992; White and Rivin, Plant Physiol. 108:831-832, 1995; Reynolds and Crawford, Plant Mol. Biol. 32:823-829, 1996). Type 1 MT-like transcripts have been detected primarily in roots (de Miranda et al., FEBS Lett. 260:277-280, 1990; Evans et al., FEBS Lett. 262:29-32, 1990; Zhou and Goldsbrough, Plant Cell 6:875-884, 1994; Hsieh et al., Plant Mol. Biol. 32:525-529, 1996) and senescent leaves (Buchanan-Wollaston, Plant Physiol. 105:839-846, 1994, Buchanan-Wollaston, Plant Mol. Biol. 33:821-834, 1997; Hsieh et al., Plant Mol. Biol. 32:525-529, 1996; Foley et al., Plant Mol. Biol. 33:583-591, 1997). Type 2 MT-like transcripts accumulate in the aerial portions such as leaves, stems, and flowers (Snowden and Gardner, Plant Physiol. 103:855-861, 1993; Foley and Singh, Plant Mol. Biol. 26:435-444, 1994; Coupe et al., Planta 197:442-447, 1995; Zhou and Goldsbrough, Mol. Gen. Genet. 248:318-328, 1995; Choi et al., Plant Physiol. 112:353-359, 1996; Whitelaw et al., Plant Mol. Biol. 33:503-511, 1997). Transcripts of type 3 MT-like genes have been detected in fruits, and show differential expression during fruit development (Ledger and Gardner, Plant Mol. Biol. 25:877-886, 1994; Lam and Abu Baker, Plant Physiol. 112:1735, 1996; and Reid and Ross, Physiologia Planatrum 100:183-189, 1997). Type 3 MT-like transcripts are also present in leaves (Dong and Dunstan, Planta 199:459-466, 1996; Bundithya and Goldsbrough, Plant Physiol. 114:S-251, 1997; Clendennen and May, Plant Physiol. 115:463-469, 1997). Some class I MT genes show programmed expression during embryogenesis. Transcripts of barley pZE40, rice Ose712 (both type 2) and white spruce EMB30 (type 3) genes are expressed temporally during embryo maturation (Smith et al., Plant Mol. Biol. 20:255-266, 1992; Chen and Chen, Plant Physiol. 114:1568, 1997; and Dong and Dunstan, Planta 199:459-466, 1996).
The invention provides, inter alia, an isolated promoter (as defined herein) from a metallothionein-like gene (i.e., the xe2x80x9cdfMTPxe2x80x9d promoter; SEQ ID NO: 17). The promoter is useful for expressing heterologous proteins either transiently in host cells or transgenically in stably transformed cells. The dfMTP promoter (SEQ ID NO: 17) can allow for developmental-specific expression of genes placed under its control.
Another aspect of the invention provides fragments and deletions of the promoter, such as those shown in SEQ ID NOS: 22, 23, 24, 25, and variants thereof. The variant promoters are characterized by their retention of at least 50% sequence identity with the disclosed promoter sequences (SEQ ID NOS: 17, 22, 23, 24, and 25), or by their retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 17, 22, 23, 24, and 25). In each case these promoters at least retain promoter activity and, in some cases, these promoters exhibit native dfMTP promoter activity.
It is also contemplated that promoters such as the CaMV35S promoter may be altered through the introduction of one or more sequences found in the dfMTP promoter. The resulting promoter is characterized by its retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 17, 22, 23, 24, and 25).
Another aspect of the invention provides vectors containing the above-described promoters and variants thereof. The vectors can be transformed into host cells. In some cases the resulting host cell can give rise to a transgenic plant.
The invention also provides transgenes. These transgenes include one of the above-described promoter sequences operably linked to one or more open reading frames (ORFs). The transgenes can be cloned into vectors and subsequently used to transform host cells, such as bacterial, insect, mammalian, fungal, yeast, or plant cells.
Accordingly, the invention provides transgenic plants such as maize, wheat, rice, millet, tobacco, sorghum, rye, barley, brassica, sunflower, seaweeds, lemna, oat, soybean, cotton, legumes, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut, and clover; lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussel sprouts, peppers and other vegetables; citrus, apples, pears, peaches, apricots, walnuts, and other fruit trees; orchids, carnations, roses, and other flowers; cacao; poplar, elms, and other deciduous trees; pine, Douglas-fir, spruce, and other conifers; turf grasses; cacao; and rubber trees and other members of the genus Hevea.
In yet another embodiment, the invention provides methods for expressing certain proteins in host cells, such as plant host cells. Such methods involve operably linking a promoter, such as a promoter as summarized above, to at least one ORF to produce a transgene, and introducing the transgene into a plant. Accordingly, the invention also provides proteins that are produced by these methods.
The promoters also can be characterized by analyzing various promoter elements found within the promoter sequence. Hence, the invention also provides promoters that maintain promoter activity and include at least eight promoter elements selected from the group consisting of E-box motifs (SEQ ID NO: 1), ERE elements (SEQ ID NO: 20), AT-rich regions (SEQ ID NO: 3), MRE elements (SEQ ID NO: 21), and ACGT core elements (SEQ ID NO: 4), and duplicates thereof, wherein the promoter displays promoter activity.
The invention also provides promoters that contain the following promoter elements in the following order: 3xe2x80x2-ERE element (SEQ ID NO: 20), AT-rich region (SEQ ID NO: 3), ERE element (SEQ ID NO: 20), ERE element (SEQ ID NO: 20), E-box motif (SEQ ID NO: 1), MRE element (SEQ ID NO: 21), ACGT core element (SEQ ID NO: 4), ACGT core element (SEQ ID NO: 4), and ACGT core element (SEQ ID NO: 4)-5xe2x80x2.
The invention also provides vectors, host cells, and transgenic plants that include a promoter as described above by their inclusion of various promoter elements.
The invention also provides methods for conferring developmental-specific expression of a gene to a plant. The method involves operably linking an ORF to a dfMTP promoter or variant there of (summarized above) to produce a transgene. The transgene is then transformed into a host cell, and the cell is regenerated into a plant.
These and other aspects of the invention will be readily apparent upon reading the following detailed description.